Leishmania are important tropical parasites, causing disease in more than 10 million people worldwide; more than 400 million people are at risk for infection in endemic regions. Currently, there are no vaccines available against leishmaniasis, and the only approved chemotherapies are marginally effective, difficult to administer, and have significant toxicity. An underlying tenet of our work is that improved understanding of key pathways required for parasite virulence and viability may provide opportunities for the development of improved therapies; several of the pathways identified in this project offer good opportunities. Leishmania are pteridine auxotrophs and our studies have revealed a fascinating and unique complexity of pteridine salvage and utilization, involving specific transporters (BT1 and FT1) and reductases (PTR1 and DHFR-TS), and now we plan to study the enzymes that utilize pteridines for the synthesis of key metabolites, determine their role in metabolism, and assess their effect on parasite virulence and survival. Our previous studies revealed for the first time a role for biopterin specifically in parasite differentiation to the infective metacyclic stage, and possibly other pathways relevant to parasite infectivity and virulence. Our aims include: 1) Studies of the Leishmania requirement for tetrahydrobiopterin (H4B) throughout the infectious cycle, and how loss of the broad spectrum pteridine reductase PTR1 leads to elevated virulence in both fly and mammalian hosts. 2) Identification of enzymes that utilize H4B as cofactors and functional dissection of their role in parasite survival and virulence, including studies of a novel aromatic amino acid hydroxylase (AAAH). Significantly, WT Leishmania synthesizes catecholamines while aaahknockouts do not, and the role of parasite-synthesized catecholamines in virulence will be sought. 3) Studies of folate metabolic enzymes such as methylene tetrahydrofolate reductase and two isoforms of methionine synthase, which play key roles in intermediary metabolism and additionally may be targets of a new class of antifolates whose action is primarily outside of their ability to inhibit DHFR and PTR1. Powerful genetic and bioinformatic screens will be used to identify new biopterin and folate utilizing enzymes and drug targets in aims 2 and 3.